Bottom Line:
In this study, we demonstrate that chemokines and HIV-1 envelope glycoproteins from both T-tropic and macrophage-tropic strains rapidly induce tyrosine phosphorylation of the protein tyrosine kinase Pyk2.The response requires CXCR4 and CCR5 to be accessible on the cell surface.The results presented here provide the first evidence for activation of an intracellular signaling event that can initiate multiple signaling pathways as a consequence of contact between HIV-1 and chemokine receptors.

ABSTRACTInfection with HIV-1 requires expression of CD4 and the chemokine receptors CXCR4 or CCR5 at the target cell surface. Engagement of these receptors by the HIV-1 envelope glycoprotein is essential for membrane fusion, but may additionally activate intracellular signaling pathways. In this study, we demonstrate that chemokines and HIV-1 envelope glycoproteins from both T-tropic and macrophage-tropic strains rapidly induce tyrosine phosphorylation of the protein tyrosine kinase Pyk2. The response requires CXCR4 and CCR5 to be accessible on the cell surface. The results presented here provide the first evidence for activation of an intracellular signaling event that can initiate multiple signaling pathways as a consequence of contact between HIV-1 and chemokine receptors.

Figure 3: Tyrosine phosphorylation of Pyk2 mediated by T-tropic gp120 can be inhibited by pertussis toxin or a monoclonal antibody against CXCR4. (a) HL60 cells were pretreated with pertussis toxin or left untreated. Treated and untreated cells were incubated with T-tropic gp120 (BH10, 5 μg/ml) for the indicated times before lysis. (b) HL60 cells were resuspended in growth media/0.5% FCS either without antibody (no Ab) or containing monoclonal antibodies specific for CXCR4 (anti-CXCR4) or human CD8 (control Ab). Cells were incubated with antibody at 37°C for 15 min, then mixed with T-tropic SF-2 envelope for 30 s before lysis.

Mentions:
To further characterize the pathway leading to Pyk2 tyrosine phosphorylation after binding of T-tropic envelope to HL60, these cells were treated with a soluble monomeric form of the gp120 subunit from the BH10 clone of the T-tropic IIIB strain (26). Tyrosine phosphorylation of Pyk2 was readily detected after addition of BH10 gp120, with kinetics similar to those observed after treatment with the chemokines SDF-1α and RANTES (Fig. 3 a, compare with Fig. 1, a and b). Gp120 is thus sufficient to stimulate Pyk2 phosphorylation, and other molecules contributed by the 293T transfectants are not required. In addition, the Pyk2 response to monomeric gp120 suggests that receptor cross-linking is not required. The Pyk2 response to BH10 gp120 was inhibitable with pertussis toxin (Fig. 3 a), implicating Gi-linked pathways in the response to either SDF-1α or HIV envelope glycoprotein in these cells.

Figure 3: Tyrosine phosphorylation of Pyk2 mediated by T-tropic gp120 can be inhibited by pertussis toxin or a monoclonal antibody against CXCR4. (a) HL60 cells were pretreated with pertussis toxin or left untreated. Treated and untreated cells were incubated with T-tropic gp120 (BH10, 5 μg/ml) for the indicated times before lysis. (b) HL60 cells were resuspended in growth media/0.5% FCS either without antibody (no Ab) or containing monoclonal antibodies specific for CXCR4 (anti-CXCR4) or human CD8 (control Ab). Cells were incubated with antibody at 37°C for 15 min, then mixed with T-tropic SF-2 envelope for 30 s before lysis.

Mentions:
To further characterize the pathway leading to Pyk2 tyrosine phosphorylation after binding of T-tropic envelope to HL60, these cells were treated with a soluble monomeric form of the gp120 subunit from the BH10 clone of the T-tropic IIIB strain (26). Tyrosine phosphorylation of Pyk2 was readily detected after addition of BH10 gp120, with kinetics similar to those observed after treatment with the chemokines SDF-1α and RANTES (Fig. 3 a, compare with Fig. 1, a and b). Gp120 is thus sufficient to stimulate Pyk2 phosphorylation, and other molecules contributed by the 293T transfectants are not required. In addition, the Pyk2 response to monomeric gp120 suggests that receptor cross-linking is not required. The Pyk2 response to BH10 gp120 was inhibitable with pertussis toxin (Fig. 3 a), implicating Gi-linked pathways in the response to either SDF-1α or HIV envelope glycoprotein in these cells.

Bottom Line:
In this study, we demonstrate that chemokines and HIV-1 envelope glycoproteins from both T-tropic and macrophage-tropic strains rapidly induce tyrosine phosphorylation of the protein tyrosine kinase Pyk2.The response requires CXCR4 and CCR5 to be accessible on the cell surface.The results presented here provide the first evidence for activation of an intracellular signaling event that can initiate multiple signaling pathways as a consequence of contact between HIV-1 and chemokine receptors.

ABSTRACTInfection with HIV-1 requires expression of CD4 and the chemokine receptors CXCR4 or CCR5 at the target cell surface. Engagement of these receptors by the HIV-1 envelope glycoprotein is essential for membrane fusion, but may additionally activate intracellular signaling pathways. In this study, we demonstrate that chemokines and HIV-1 envelope glycoproteins from both T-tropic and macrophage-tropic strains rapidly induce tyrosine phosphorylation of the protein tyrosine kinase Pyk2. The response requires CXCR4 and CCR5 to be accessible on the cell surface. The results presented here provide the first evidence for activation of an intracellular signaling event that can initiate multiple signaling pathways as a consequence of contact between HIV-1 and chemokine receptors.